Cd8alphadendritic cell differentiated from human hematopoietic stem cell and a method for differentiation

ABSTRACT

Lymphoid dendritic cells with an immunophenotype of CD8α+ can be produced by a method which comprises culturing human hematopoietic stem cells in a medium containing GM-CSF, followed by culturing in a medium containing IFN-Υ, said cells being useful in various immunotherapies.

FIELD OF THE INVENTION

[0001] The present invention relates to lymphoid dendritic cellsdifferentiated from human hematopoietic stem cells, a method fordifferentiating the lymphoid dendritic cells from human hematopoieticstem cells and a pharmaceutical composition for immunotherapy containingthe lymphoid dendritic cells as an active ingredient.

BACKGROUND OF THE INVENTION

[0002] Dendritic cells (DCs) are powerful antigen presenting cells(APCs)that orchestrate various immune responses against specific antigens andat the same time suppress auto-immune response by deleting potentiallyautoreactive T cells. These apparently contradictory functions have beensuggested to originate from different subsets of DCs.

[0003] Murine DCs can be subdivided into at least two distinct subtypes,myeloid and lymphoid DCs, on the basis of their anatomic localization,transplantation experiments, and cell surface phenotypes. DCs bearingthe CD11c⁺, MHCII⁺, CD4⁺, CD8α⁻ cell surface phenotype, called CD8α⁻myeloid DCs, can be derived from myeloid precursor cells, whereas DCsbearing the CD11c⁺, MHCII⁺, CD4⁻, CD8α⁺ cell surface phenotypes, calledCD8α⁺ lymphoid DCs, are present in thymus or spleen.

[0004] It has been reported that murine CD8α⁺ lymphoid DCs also produceIFN-γ in response to IL-12 like NK cells(natural killer cells) and Tcells (Toshiaki et al., Brief Definitive Report, 189(12),1981-1986(1999)), Also, when lymphoid DCs such as murine Langerhanscells are injected into CD8α⁻ mice, they are transferred into the lymphnode and differentiate into CD8α⁺ DCs and the differentiated CD8α⁺ DCscan produce IFN-γ (Miriam et al., Blood, 96(5), 1865-1872(2000)).

[0005] Such lymphoid DCs not only induce immune tolerance but functionas powerful immunogenic APCs against various allogeneic antigens,activating T helper cell type I response by producing IL-12 and IFN-γ.

[0006] DCs are present virtually in all tissues of the body, but in lowconcentrations, and it is therefore difficult and cumbersome to procurea sufficient amount of DCs for ex vivo manipulation.

[0007] Accordingly, various efforts have been made to generate DCs exvivo from HSCs or monocytes in a large scale, using for example, severalcytokines including Granulocyte-macrophae colony stimulating factor(GM-CSF), interleukin-4(IL-4), tumor necrosis factor-alpha (TNF-α) andstem cell factor (SCF). DCs which express CD1a⁺, CD4⁺, CD11c⁺, CD40⁺,CD54⁺, CD80⁺, CD83⁺, CD86⁺, HLA class I⁺, HLA class II⁺, CD3⁻, CD8⁻ andCD14⁻ were generated by using GM-CSF (Williams et al., Int. Rev. Cytol.,153: 412(1994); Santiago-Schwarz et al., Nature, 360: 258(1993); andRosenzwajg et al., Blood, 87: 535(1996)).

[0008] However, it has never been reported that DCs of CD8α⁺immunophenotype (CD8α⁺ DCs) are present in human or that CD8α⁺ DCs canbe differentiated from human hematopoietic stem cells(HSCs).

SUMMARY OF THE INVENTION

[0009] Accordingly, it is a primary object of the present invention toprovide a lymphoid dendritic cells of a CD8α⁺ immunophenotype which aredifferentiated from human hematopoietic stem cells.

[0010] It is another object of the present invention to provide a methodfor differentiating the lymphoid dendritic cells from humanhematopoietic stem cells.

[0011] It is still another object of the present invention to provide apharmaceutical composition for immunotherapy comprising the lymphoiddendritic cells.

[0012] It is a further object of the present invention to provide amethod for treating an immune-related disease in a mammal.

[0013] In accordance with one aspect of the present invention, there isprovided lymphoid dendritic cells of CD8α⁺ immunophenotype of which aredifferentiated from human hematopoietic stem cells.

[0014] In accordance with another aspect of the present invention, thereis provided a method for differentiating the lymphoid dendritic cellswhich comprises culturing human hematopoietic stem cells in two steps,first in a first medium containing GM-CSF and then in a second mediumcontaining IFN-γ.

[0015] In accordance with another aspect of the present invention, thereis provided a pharmaceutical composition for immunotherapy whichcomprises a therapeutically effective amount of the dendritic cells.

[0016] In accordance with still another aspect of the present invention,there is provided a method for treating an immune-related disease in amammal, which comprises administering the lymphoid dendritic cells to asubject in need thereof in an amount effective for treating the disease.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The above and other objects and features of the present inventionwill become apparent from the following description of the inventiontaken in conjunction with the following accompanying drawings, wherein:

[0018]FIGS. 1A, 1B and 1C: light microscope(A), scanning electronmicroscope(B) and transmission electron microscope(C) photographs oflymphoid dendritic cells differentiated from human hematopoietic stemcells;

[0019]FIG. 2: the culture time-dependent change of the dendritic cellcount;

[0020] FIGS. 3A and 3B: histograms showing the immunophenotye of thelymphoid dendritic cells (A: a cell after culturing for 7 days; B: acell after culturing for 14 days; x axis: cell count; y axis:fluorescent intensity; open histogram: a negative control; and filledhistogram: a positive cell group for cell surface antigen);

[0021] FIGS. 4A and 4B: histograms showing the immuno phenotye of thelymphoid dendritic cells(A: only CD3 and CD4 are stained, and B: onlyCD3 and CD8α are stained);

[0022]FIGS. 5A, 5B and 5C: the ability to phagocytize FITC-labeleddextran (A: a control; B: a cell group after culturing for 1 week; C: acell group after culturing for 2 weeks; open histogram: a negativecontrol and filled histogram: a cell group which phagocytizeFITC-labeled dextran);

[0023]FIG. 6: the ability to stimulate T-cell proliferation (♦: a cellgroup after culturing for 1 week and ▪: a cell group after culturing for2 weeks);

[0024] FIGS. 7A and 7B: ELISA(enzyme-linked immunosorbant assay) resultsshowing IL-12(A) and IFN-γ (B) producing ability of CDs (1: the amountof protein produced after culturing for 1 week using GM-CSF; 2: theamount of protein produced after culturing for 1 week using GM-CSF,followed by culturing for 1 week using IFN-γ; and 3: the amount ofprotein produced after culturing for 1 week using GM-CSF and for 1 weekusing IFN-γ, followed by culturing for 1 day with added ionomycin.

DETAILED DESCRIPTION OF THE INVENTION

[0025] The CD8α⁺ lymphoid dendritic cells of the present invention haveimmunophenotypes of CD1a⁻, CD3⁻, CD4⁻, CD8⁺, CD11c⁺, CD14⁻, CD40⁺,CD54⁺, CD80⁺, CD83⁺, CD86⁺, HLA-I⁺ and HLA-II⁺.

[0026] The CD8α⁺ lymphoid dendritic cells can be prepared by culturinghematopoietic stem cells in two steps, first in a first mediumcontaining GM-CSF (granulocyte macrophage-colony stimulating factor) foran appropriate period, e.g., 3 to 9 days, and then in a second mediumcontaining IFN-γ (interferon-γ), e.g., for 3 to 9 days.

[0027] In the inventive process, GM-CSF and IFN-γ may be added to themedium every 2-4 days, preferably 3 days, in amounts ranging from 1 to1,000 ng/ml and from 1 to 1,000 U/ml, respectively, preferably, 20 to200 ng/ml and 50 to 500 U/ml, respectively.

[0028] In order to enhance the differentiation of mature DCs, it ispreferred to add ionomycin, lipopolysaccharide (LPS) or keyhole limpethemocyanin (KLH) to the second medium in an amount of 0.1 to 10 μg/ml,preferably 1 μg/ml, on the later part of the second stage culture andculture until the end of the second culture period, preferably for 1day.

[0029] The CD8α⁺ lymphoid dendritic cells of the present invention haveadvantages in that: interleukin-12 and IFN-γ can be produced in highyields; T cells can be stimulated into proliferation; and powerfulcellular immune responses can be induced by activating T helper cellsand cytotoxic T lymphocyte.

[0030] The CD8α⁺ lymphoid dendritic cells of the present invention canbe used as an active ingredient of a pharmaceutical compostion for animmunotherapy for a immune-related disease. Non-limiting examples ofimmune-related diseases, which can be treated by using the CD8α⁺lymphoid dendritic cell of the present invention, include any kind ofmalignant diseases, tuberculosis infections, HIV infections andauto-immune diseases.

[0031] A pharmaceutical composition for preventing or treating immune-related diseases can be prepared by mixing the inventive CD8α⁺ lymphoiddendritic cells with a pharmaceutically acceptable excipient or carrier,or by diluting it with a pharmaceutically acceptable diluent inaccordance with any of the conventional procedures. Examples of suitablecarriers, excipients, and diluents are lactose, dextrose, sucrose,sorbitol, water, and mineral oil. The formulations may additionallyinclude fillers, anti-agglutinating agents, preservatives and the like.The pharmaceutical composition of the present invention may beformulated so as to provide quick, sustained or delayed release of theactive ingredient after their administration to a mammal by employingany of the procedures well known in the art. Thus, the formulations maybe in the form of a sterile injectable solution, suspension, emulsion,solution and the like, wherein a sterile injectable solution ispreferred.

[0032] Accordingly, the present invention also provides a method oftreating an immune-related disease in a mammal, which comprisesadministering the inventive CD8α⁺ lymphoid dendritic cells to a subjectin need thereof in an amount effective for treating the disease.

[0033] The inventive CD8α⁺ lymphoid dendritic cells may be administeredto a patient by the conventional immunotherapy methods. Specifically,auto-cells are taken from the patient and cultured to obtain DCs havingan immunological enhancement effect and the DCs are pulsed with a targetantigen. In this process, in order to increase the immunologicalenhancement effect of DCs, the DCs may be pulsed in the presence ofradiation or ultraviolet-treated cancer cells, a lysate of cancer cellskilled by freezing-thawing or a cytotoxic drug. Another method is topulse DCs using a DNA, RNA, protein or peptide as an antigen. The DCspulsed with a specific antigen may be directly injected into a patient,or T cells activated by the inventive DCs may be injected. Further, toenhance therapeutic effects, it is preferred to inject the inventive DCstogether with IL-2.

[0034] The cell composition of the present invention can be administeredvia various routes including transdermal, subcutaneous, intravenous andintramuscular introduction, and direct injection into cancerous regions.

[0035] Typical unit dose of the CD8α⁺ lymphoid dendritic cells may rangefrom 1×10⁷ to 1×10⁹ cells and they can be administered weekly or monthlyfor 6 months. However, it should be understood that the amount of theactive ingredient actually administered ought to be determined in lightof various relevant factors including the disease to be treated, theseverity of the patient's symptom, the chosen route of administration,and the age, sex and body weight of the individual patient; and,therefore, the above dose should not be intended to limit the scope ofthe invention in any way.

[0036] The immunotherapy using the inventive pharmaceutical compositionis advantageous in that the immunologic rejection does not occur due tothe use of the dendritic cells differentiated from the patient'shematopoietic stem cells and also in that the dendritic cells injectedinto the body can continuously produce cytokines.

[0037] The inventive pharmaceutical composition induces a strongcellular immuno-response against diseases caused by specific antigens,stimulating T cell proliferation, and it can be advantageously used inanti-cancer and anti-virus therapies.

[0038] The following Examples are intended to further illustrate thepresent invention without limiting its scope; and the experimentalmethods used in the present invention can be practiced in accordancewith Examples given herein below, unless otherwise stated.

[0039] Further, percentages given below for solid in solid mixture,liquid in liquid, and solid in liquid are on the bases of wt/wt, vol/voland wt/vol, respectively, unless specifically indicated otherwise.

EXAMPLE 1 Extraction of Hematopoietic Stem Cell and Generation ofDendritic Cell

[0040] In order to mobilize peripheral blood stem cells (PBSCs),granulocyte colony stimulating factor (G-CSF, Lenograstim, chugai, Co.,Tokyo, Japan) were injected subcutaneously into 10 patients with variousneoplastic diseases (breast cancer, leukemia and lymphoma) at a dose of300 μg/day, respectively. Peripheral blood stem cells were extracted inaccordance with leukapheresis using Cobe Spectra (Cobe BCT, Inc.,Lakewood, Colo., USA) cell separator on day 4 after in the injection.Mononuclear cells were separated from the peripheral blood stem cellsthus obtained in accordance with the density gradient centrifugationmethod using Ficoll-Hypaque (Histopaque, Sigma Chemical, St. Louis, Mo.,USA) and washed twice with phosphate buffered saline (PBS, SigmaChemical). The mononuclear cells filtered with a 30 μm nylon meshmembrane in PBS containing 5% bovine serum albumin (BSA). Mononuclearcells were collected, added FcR blocking reagent, reacted with CD34microbeads (Miltenyi Biotec GmbH) at 4° C. for 30 minutes, washed withPBS containing BSA, passed cells through mesh and CD34⁺ mononuclearcells were separated by conducting high gradient immunomagneticseparation (HGIS; MidiMACS, Miltenyi biotech, USA). 1×10⁵/ml of theseparated CD34⁺ peripheral blood stem cells were cultured in X-VIVO 20medium (Biowhittaker, Walkersville, Md., USA) with 5% human serumalbumin, 100 U/ml of penicillin, 100 μg/ml of streptomycin (Sigma), 100U/ml of L-glutamine (Sigma) for 2 weeks, while adding 50 ng/ml of GM-CSF(Leucogen, LG Chemical Co., Korea) every three days during the firstweek, followed by adding 200 U/ml of IFN-γ (Intermax-γ, LG Chemical Co.,Korea) every three days during the second week. On day 13, i.e., the daybefore the end of the second week, 1 μg/ml of ionomycin (Sigma) wasadded to the medium and cultured for 1 day to allow the cells to mature.

[0041] As shown in FIG. 1, the cultured cells have relatively abundantcytoplasm with multiple dendrites (Giemsa-Wright stain). The cellscolonized and conglomerated on the surface of the culture flask duringthe early phase of culturing but started to separate and proliferatewhen IFN-γ was added on the second week of culturing. Further, as can beseen in the scanning and transmission electron micrographs, the cellsreveal the DCs' characteristic features: the nucleus is shifted to oneside of the region; multiple dendrites are present on the cell surface;and the abundant cytoplasm has many granules.

[0042] On the other hand, in order to examine the culture time-dependantenhancement in the dendritic cell count, 1 ml samples of the culturesolutions after 1 week and 2 weeks were examined with hemocytometer.

[0043] As shown in FIG. 2, the total cell count increased continuouslythroughout the culture period, especially after adding IFN-γ. On day 14,the last day of culturing, the cell count was about 30×10⁵/ml, about 30times higher than the initial cell count.

EXAMPLE 2 Examination of Immunophenotype of the Differentiated DCs

[0044] To confirm the immunophenotype of the cultured DCs, 1×10⁵ ofcells were reacted with fluorescein isothiocyanate orphycoerythrin-labeled specific monoclonal antibodies for CD1a, CD3, CD4,CD8α, CD11c, CD14, CD80, CD83, CD86, HLA class I(ABC), and HLA classII(DR) (Pharmingen, San diego, Calif., USA) in PBS containing 5% FBS(fatal bovine serum) at room temperature for 15 minutes in the darkroom, and the resulting solution was washed with PBS, and then analyzedby flow cytometry (FACScan, Becton Dickinson).

[0045] As shown in the FIGS. 3A and 3B, on day 7 of the culture, thecells expressed CD1a, CD11c as well as CD40, CD54, CD80, CD86 and HLAclass I/II. However, CD83⁺, a maker for mature DCs, expressed weakly.The addition of ionomycin altered the CD1a and CD14 into the negativephenotypes and the cells of CD83⁺ phenotype increased. Further, as shownin the FIGS. 4A and 4B, the cells exhibited negative phenotypes of CD3and CD4, while CD8α phenotype was positive.

[0046] These results suggested that the lymphoid dendritic cellsdifferentiated from human hematopoietic stem cells are lymphoid DCs thatexpress CD1a⁻, CD3⁻, CD4⁻, CD8α⁺, CD11c⁺, CD14⁻, CD40⁺, CD54⁺, CD80⁺,CD83⁺, CD86⁺, HLA-I⁺ AND HLA-II⁺ and, most interestingly, CD8α⁺.

EXAMPLE 3 Confirmation of the Phagocytic Ability of CD8α⁺ DCs

[0047] To confirm the phagocytic ability of the differentiated DCs,2×10⁵ of DCs was mix-incubated with dextran-FITC (Sigma) at 37° C. for 1hour without FBS. Then, the dextran engulfed cells were examined by aflow cytometry.

[0048] As shown in the FIGS. 5B and 5C, CD8α⁺ CDs had high phagocyticactivity. Further, on day 14 of the culture, the phagocytic activity wasfurther augmented. A control experiment at 4° C. (5A) showed theobserved phagocytic activity of the inventive CD8α⁺ CDs is real, not anartifact created by measurement conditions.

EXAMPLE 4 Examination of Inducting Ability of CD8α⁺ DCs in LymphocyteProliferation

[0049] To examine the ability of DCs in stimulating T cellproliferation, allogeneic mixed lymphocyte reaction (MLR) was carriedout as follows.

[0050] Peripheral mononuclear cells (MNCs) were isolated from normalvolunteer's blood in accordance with the density gradient centrifugationmethod using Ficoll-Hypaque (Histopaque; Sigma Chemical, St. Louis, Mo.,USA) and washed with PBS. 3×10⁸ of MNC thus obtained were incubated in ahuman T cell enrichment column (R & D, USA) at room temperature for 10minutes and T cells were extracted with PBS. Then, γ-ray irradiated(30Gy) DCs and T cells (effector:responder) were added to the microplate invarying ratios of 1:1, 1:10, 1:10², 1:10³ and 1:10⁴, in triplicates, andthen, incubated at 37° C. for 3 days. 20 μl ofBrdU(5-bromo-2′-deoxyuridine) was added thereto and further incubatedfor 24 hours. Formaldehyde was added to the plate and reacted at roomtemperature for 30 minutes to fix the cells, 100 μl of an anti-BrdUsolution was added to the plate, and allowed to react at roomtemperature for 90 minutes. Then, the plate was washed with PBS andreacted with a color development-substrate for 30 minutes. 1N H₂SO₄ wasadded thereto to stop the reaction and the absorbance at 450 nm wasmeasured with ELSA plate reader (Molecular device, USA).

[0051] As the result in FIG. 6 shows, a burst of T cell proliferationwas observed at a ratio of 1:10⁴ (effector:responder). This demonstratesthat the lymphoid DCs are functionally capable of stimulating theproliferation of the T lymphoid.

EXAMPLE 5 Determination of Cytokine Release of Lymphoid DCs

[0052] The cells' cytokine releasing capability was measured byELISA(enzyme-linked immunosorbant assay) as follows.

[0053] An anti-human IL-12 and IFN-γ antibodies(Pharmingen) were dilutedin 0.1 mol/l of NaHCO₃ to 2 μg/ml, and 50 μl of the resulting solutionwas divided to the ELISA plate(Corning) to be incubated at 4° C. for 24hours. PBS containing 5% FBS was added to block the plate for 3 hours.50 μl of a standard sample and 50 μl each of the culture solutions atday 7, 13 and 14 of the culture were added thereto and reacted for 4hours.

[0054] 2 μg/ml of Biotin-conjugated detection antibody was added to theplate and reacted for 3 hours. The plate was washed, treated with astreptavidin-horseradish peroxidase solution (diluted to 1:2000) for 1hour, washed again, and then treated with TMB (Zymed, San Francisco,Calif., USA), followed by measuring the absorbance at 450 nm using ELISAmicroplate reader(Molecular device, USA).

[0055] As the results in the FIGS. 7A and 7B show, the amounts ofcytokines produced were very low until day 13, but jumped to high levelsafter ionomycin was added.

[0056] While the invention has been described with respect to the abovespecific embodiments, it should be recognized that various modificationsand changes may be made to the invention by those skilled in the artwhich also fall within the scope of the invention as defined by theappended claims.

What is claimed is:
 1. A lymphoid dendritic cell of CD8α⁺immunophenotype of which is differentiated from human hematopoietic stemcells.
 2. The lymphoid dendritic cell of claim 1 having CD1a⁻, CD3⁻,CD4⁻, CD8⁺, CD11c⁺, CD14⁻, CD40⁺, CD54⁺, CD80⁺, CD83⁺, CD86⁺, HLA-I⁺ andHLA-II⁺ immunophenotypes.
 3. The lymphoid dendritic cell of claim 1which is capable of inducing the production of interleukin-12 (IL-12)and interferon-γ (IFN-γ).
 4. A method for differentiating the lymphoiddendritic cell of claim 1 which comprises culturing human hematopoieticstem cells in two steps, first in a first medium containing GM-CSF andthen in a second medium containing IFN-γ.
 5. The method of claim 4,wherein GM-CSF and IFN-γ are added to the respective mediums every 2-4days in amounts of 1 to 1,000 ng/ml and 1 to 1,000 U/ml, respectively.6. The method of claim 4, which further comprises a step of adding 0.01to 10 μg/ml of ionomycin, lipopolysaccharide (LPS) or keyhole limpethemocyanin (KLH) to the second medium during the course of the secondculture.
 7. The method of claim 4, wherein the immunophenotype of thelymphoid dendritic cell is CD1a⁻, CD3⁻, CD4⁻, CD8α⁺, CD11c⁺, CD14⁻,CD40⁺, CD54⁺, CD80⁺, CD83⁺, CD86⁺, HLA-I⁺ and HLA-II⁺.
 8. The method ofclaim 4, wherein the lymphoid dendritic cell is capable of inducing theproduction of interleukin-12(IL-12) and interferon-γ (IFN-γ).
 9. Apharmaceutical composition for immunotherapy which comprises atherapeutically effective amount of the dendritic cell of any one ofclaims 1 to
 3. 10. The pharmaceutical composition of claim 9, whereinthe immunotherapy is an anti-cancer or an anti-virus therapy.
 11. Amethod for treating an immune-related disease in a mammal, whichcomprises administering the lymphoid dendritic cell of any one of claims1 to 3 to a subject in need thereof in an amount effective for treatingthe disease.